Mitochondrial Dysfunction and Adipose Insulin Resistance Abstract A variety of animal, cell culture and molecular studies have correlated increased oxidative stress and the accumulation of reactive oxygen species (ROS) such as superoxide anion and hydroxyl radicals to type 2 diabetes. While appreciated for years, it was unclear if oxidative stress / ROS was a casual or causal factor in the etiology of the disease. However, more recent work utilizing a variety of loss and gain of function analyses have indicated that oxidative stress is causally linked to insulin resistance but that the molecular mechanisms remain obscure. This application will profile new and compelling findings from our laboratories using animal and cell culture models that establish a molecular linkage between the antioxidant defense system in adipose cells with mitochondrial function, oxidative phosphorylation, signal transduction and the development of insulin resistance. We present a novel hypothesis supported by preliminary studies from genomic, proteomic, metabolomic and molecular analyses that point toward the covalent modification of mitochondrial proteins with bioactive lipids and the oxidation of mitochondrial thioredoxin as central to the process. Initiating this oxidative stress challenge are new findings that describe the tumor necrosis factor [unreadable] (TNF[unreadable]) dependent down regulation of glutathione S-transferase A4 setting the stage for a molecular cascade of events that activates the c-JUN NH2-terminal kinase (JNK), an established regulator of insulin sensitivity. Moreover, we present new findings that demonstrate that the down regulation of GSTA4 is not merely a process observed in animal models of insulin resistance but also occurs selectively in obese, insulin resistant, but not obese, insulin sensitive humans thereby providing a molecular differentiation between obesity and insulin resistance. This application builds on recent evidence obtained in the Bernlohr, Griffin and Arriaga laboratories that functionally links oxidative stress to insulin resistance. These studies in sum lead to our central hypothesis: decreased expression of GSTA4 in adipocytes leads to increased mitochondrial protein carbonylation. Carbonylation of mitochondrial protein results in decreased TCA cycle activity, defective State 3 respiration and increased superoxide anion production. Mitochondrial dysfunction and ROS production leads in turn to the relative oxidation of Thioredoxin 2 (Trx2) and the activation of Trx2-ASK1- JNK/p38 signaling system contributing to insulin resistance. To test this hypothesis, the following four specific aims are proposed: Aim 1. Identify and evaluate mitochondrial protein carbonylation in cell culture and animal models. Aim 2. Assess mitochondrial function and ROS production in cell culture and animal models. Aim 3. Develop and characterize aP2-HA-GSTA4 transgenic mice maintained on low and high fat diets. Aim 4. Characterize the regulation of insulin signaling by the Trx2-ASK1-JNK pathway in animal and cell culture models.